We generated an integrating， CD46-targeted， helper-dependent adenovirus HDAd5/35++ vector system for hematopoietic stem cell (HSC) gene therapy. The ∼12-kb transgene cassette included a β-globin locus control region (LCR)/promoter driven human γ-globin gene and an elongation factor alpha-1 (EF1α)-mgmt expression cassette， which allows for drug-controlled increase of γ-globin-expressing erythrocytes. We transduced bone marrow lineage-depleted cells from human CD46-transgenic mice and transplanted them into lethally irradiated recipients. The percentage of γ-globin-positive cells in peripheral blood erythrocytes in primary and secondary transplant recipients was stable and greater than 90%. The γ-globin level was 10%-20% of adult mouse globin. Transgene integration， mediated by a hyperactive SB100x transposase， was random， without a preference for genes. A second set of studies was performed with peripheral blood CD34 cells from mobilized donors. 10 weeks after transplantation of transduced cells， human cells were harvested from the bone marrow and differentiated into erythroid cells. Erythroid cells expressed γ-globin at a level of 20% of adult α-globin. Our studies suggest that HDAd35++ vectors allow for efficient transduction of long-term repopulating HSCs and high-level， almost pancellular γ-globin expression in erythrocytes. Furthermore， our HDAd5/35++ vectors have a larger insert capacity and a safer integration pattern than currently used lentivirus vectors.